MTOR Inhibition With Rapamycin Mitigates Radiation-Induced Pulmonary Fibrosis in a Murine Model

Abstract

Fibrosis is a late toxicity of radiation in lung. Radiation-induced pulmonary fibrosis (RIPF) is characterized by parenchymal cell depletion, inflammation, fibroblast proliferation, and collagen deposition. mTOR signaling has been implicated in inflammatory cytokine production, fibroblast proliferation, and epithelial stem cell senescence. We sought to determine if mTOR inhibition with rapamycin would mitigate RIPF. C57Bl/6 mice received a diet with rapamycin (14 mg/kg food; 0.056 mg/mouse/day) or control diet beginning one day prior to radiation and continuing until tissue collection or death. Cohorts of mice treated with rapamycin or control diet were exposed to thoracic irradiation in 5 daily fractions of 6 Gy (RT), and followed for survival (n=8 per group) and tissue collection (n=3 per time point). Histologic changes were evaluated by Masson-Trichrome staining at 16 weeks after RT. Inflammatory cytokine expression was evaluated by quantitative real time PCR. Senescence was assessed by staining for beta-galactosidase activity. To define the role of rapamycin in the fibroproliferative response, cell proliferation (MTT assay) and collagen deposition (Hydroxyproline assay) were examined in a mouse fibroblast cell line. Administration of rapamycin increased the median survival of irradiated mice from 116 days to 156 days (log rank p=0.006). At 16 weeks after irradiation, treatment with rapamycin reduced hydroxyproline content compared to vehicle treated irradiated controls (RT+vehicle: 45.9±11.8, RT+rapamycin: 21.4±6.0, or unirradiated+control chow: 25.7±5.0 μg/lung respectively, p=0.001). Mice exposed to RT+vehicle had dense foci of subpleural fibrosis at 16 weeks, a finding largely absent in RT+rapamycin treated mice. Rapamycin treatment attenuated IL-1β and TGF-β induction in lung in response to RT at multiple time points. Cellular senescence was significantly decreased by rapamycin treatment in primary type 2 pneumocytes (4-fold reduction at 5 days after RT, p=0.012) and in lung tissue at 16 weeks after RT (3-fold decrease, p<0.001). Treatment of NIH3T3 fibroblasts with rapamycin (50 ng/ml) resulted in decreased collagen production (1.3-fold decrease at 3 days after RT, p=0.013) and decreased proliferation (1.2-fold decrease, p=0.019). Rapamycin protected against RIPF in murine lungs through a reduction in inflammatory cytokine expression, extra cellular matrix production, and senescence in type2 pneumocytes. Rapamycin has the potential to be an effective therapy option for pulmonary fibrosis following thoracic radiation.